Soil bacteria of the genus Rhizobium, a member of the family Rhizobiaceae, are capable of infecting plants and inducing a highly differentiated structure, the root nodule, within which atmospheric nitrogen is reduced to ammonia by the bacteria. The host plant is most often of the family Leguminosa. Previously, Rhizobium species were informally classified in two groups, either as "fastgrowing" or "slow-growing" to reflect the relative growth rates in culture. The group of "slow-growing" rhizobia has recently been reclassified as a new genus, Bradyrhizobium (Jordan, D.C. (1982) Int. J. Syst. Bact. 32:136). The fast-growing rhizobia include Rhizobium trifolii, R. meliloti, R. leguminosarum and R. phaseolus. These strains generally display a narrow host range. Fast-growing R. japonicum which nodulate wild soybeans, Glycine max cv. Peking and siratro, and fast-growing members of the cowpea Rhizobium display broader host range. R. japonicum strains form only ineffective nodules on commercial soybean cultivars. The slow-growing rhizobia, now a distinct genus called Bradyrhizobium, include the commercially important soybean nodulating strains Bradyrhizobium japonicum (i.e., USDA 110 and USDA 123), the symbiotically promiscuous rhizobia of the "cowpea group," and Bradyrhizobium sp. (Parasponia) (formerly Parasponia Rhizobium) which nodulates the nonlegume Parasponia, as well as a number of tropical legumes including cowpea and siratro.
Nodulation and development of effective symbiosis is a complex process requiring both bacterial and plant genes. The symbiotic association between bacteria of the genus Bradyrhizobium or Rhizobium and leguminous plants results from a set of both temporally and spatially defined interactions between the plant host and the microorganism. These interactions are mediated through the expression of a number of bacterially-encoded and plant-encoded genes (Vincent, J.M. (1977) in Rhizobium General Microlrology. A Treatise on Dinitrooen Fixation Section III, R.W.F. Hardy and W.S. Silver (eds.), Wiley, N.Y., pp. 277-366). The temporal coordination of the early steps of nodule formation is achieved, at least in part, through the induction of bacterial nodulation gene expression by low molecular weight signal molecules from the host (Redmond et al. (1986) Nature 323:632-636; Peters et al. (1986) Science 223:977-979) and the reciprocal requirement for the expression of certain bacterial gene products prior to the induction of host gene expression (Govers et al. (1986) Nature 323:564-566).
Microscopy studies have shown that the infection process begins with attachment of bacteria to the root hair surface, induction of a root hair curling response and infection thread formation. This thread then grows down through the root hair cell into cortical tissue. For nodules with a determinate structure, cell division is induced in the pericycle and cortical tissue of the root at an early stage of infection. The infection thread continues to grow and ramifies within the expanding cortical zone. Bacteria bud off from the infection threads and form bacteroides surrounded by a plant-derived membrane, and nitrogen fixation occurs within these structures (Bauer, W.D. (1981) Ann. Rev. Plant Physiol. 32:407-449). Studies of the timing of nodule development using spot inoculation assays have shown that under these conditions, the first events of root hair curling and the induction of cell divisions within the cortex and pericycle of the root occur within 24-48 hours after inoculation with bacteria (Calvert et al. (1984) Can. J. Bot. 62:2375-2384; Ridge, R.W. and Rolfe, B.G. (1986) J. Plant Physiol. 122:121-137).
Several recent reviews of the genetics of the Rhizobium-legume interaction are found in Broughton, W.J., ed. (1982) Nitrogen Fixation, Volumes 2 and 3 (Clarendon Press, Oxford); Puhler, A. (ed.) (1983) Molecular Genetics of the Bacteria-Plant Interaction (Cornell University Publishers, Ithaca, N.Y.); Long, S.R. (1984) in Plant Microbe Interactions Volume 1, Kosuge, T. and Nester, E.W. (eds.), McMillan, N.Y., pp. 265-306; and Verma, D.P.S. and Long, S.L. (1983) International Review of Cytology (Suppl. 14), Jeon, K.W. (ed.), Academic Press, p. 211-245.
In the fast-growing species, the genes required for nodulation and nitrogen fixation are located on large Sym (symbiotic) plasmids. Although the process of recognition, infection and nodule development is complex, it appears that at least for the fast-growing rhizobia relatively few bacterial genes are directly involved and these are closely linked on the Sym plasmid. For example, a 14 kb fragment of the Rhizobium trifolii Svm plasmid is sufficient to confer clover-specific nodulation upon a Rhizobium strain cured of its Svm plasmid, as well as on an Aorobacterium strain which does not normally nodulate plants (Schofield et al. (1984) Plant Mol. Biol. 3:3-11). Nodulation and nitrogenase genes are localized on symbiotic plasmids in R. leguminosarum (Downie et al. (1983) Mol. Gen. Genet. 190:359-365) and in R. meliloti (Kondorosi et al. (1984) Mol. Gen. Genet. 193:445-452).
Fine structure genetic mapping has been used to locate individual nodulation genes in fast-growing rhizobia. Transposon mutagenesis, most often using the transposon Tn5, has identified about 10 nodulation genes associated with non-nodulation, delayed nodulation and altered host range phenotypes (Djordjevic et al. (1985) Mol. Gen. Genet. 200:263-271; Downie et al. (1985) Mol. Gen. Genet. 198:255-262; Kondorosi et al. (1984) Mol. Gen. Genet. 193:445-452; Innes et al. (1985) Mol. Gen. Genet. 201:426-432; Kondorosi et al. (1985) Nitrooen Fixation Research Progress, Evans et al. (eds.) Martinus Nijhoff, Dordrecht, Netherlands, pp. 73-78; Long et al. (1985) ibid., pp. 87-93; Downie et al. (1985) ibid. pp. 95-100; Rolfe et al. (1985) ibid., pp. 79-85; Schofield and Watson (1985) ibid., p. 125).
Three "common" Sym plasmid encoded nodulation genes have been identified in R. meliloti (Torok et al. (1984) Nucl. Acids Res. 12:9509-9524; Jacobs et al. (1985) J. Bacteriol. 162:469-476), R. leguminosarum (Rossen et al. (1984) Nucl. Acids Res. 12:9497-9508) and R. trifolii (Schofield and Watson (1986) Nucl. Acids Res. 14:2891-2903; Rolfe et al. (1985) Nitrogen Fixation Research Prooress, Evans et al. (eds.), Martinus Nijhoff, p. 79-85; Schofield and Watson, ibid., p. 125; Schofield Ph.D. Thesis (1984) Australian National University, Canberra, Australia). These genes, designated nodA, B and C, are associated with the early stages of infection and nodulation and are functionally and structurally conserved among fast-growing rhizobia. In R. meliloti, R. leguminosarum and R. trifdii, the nodA, B and C genes are organized in a similar manner and are believed to be coordinately transcribed as a single genetic operon. Mutations in these genes fail to develop visible nodules (Nod-) and in some cases fail to induce root hair curling which is prerequisite for infection.
The DNA region adjacent to nodA (5'- from the start of nodA) in R. meliloti was also reported to be involved in early nodulation function (Torok et al. (1984)supra). The region adjacent to the nodABC operon in fast-growing rhizobia has now been shown to contain an open reading frame, now designated nodD. The location and sequence of nodD has been determined in R. meliloti (Eglehoff et al. (1985) DNA 4:241-248), R. leguminosarum (Sherman et al. (1986) EMBO J. 5:647-652; Downie et al. (1985) Mol. Gen. Genet. 198:255-262) and R. trifolii (Schofield and Watson (1985) supra and 1986; Rolfe et al. 1985; Schofield, 1984) supra. Mutations in nodD of R. meliloti have been shown to be functionally complemented by the nodD gene of R. trifolii (Fisher et. al. (1985) Appl. Environ. Microbiol. 49:1432-1435Comparison of the sequences of nodD genes in the fast-growing rhizobia confirm that there is significant sequence conservation. R japanicum USDA 191, a promiscuous, fast-growing rhizobium, is also found to contain two distinct nodD-like genes, nodD-r.sub.1 and nodD-r.sub.2 (EPO Publication No. 0211662, 1987) These nodD-like genes are about 70% homologous to each other and both display about the same homology to nodD genes of other fast-growing strains. The structural conservation of the nodD genes confirms that these genes function similarly in the different strains.
In contrast to the fast-growing rhizobia, no Sym plasmids have been associated with nodulation by the slowgrowing rhizobia, B. japonicum or Bradyrhizobium sp. (Parasponia). The nitrogenase and nodulation genes of these organisms are believed to be encoded on the chromosome. Marvel et al. (1984) in Advances in Nitrogen Fixation Research, Veeger and Newton (ed.) Nijhoff/Junk, the Hague, Netherlands; and (1985) Proc. Natl. Acad. Sci. 82:5841-5845, have shown that a strain of Bradyrhizobium sp. (Parasponia) contains genes associated with early nodulation, which can functionally complement mutations in R. melilati nod gene mutants and which hybridize to the nodABC genes of R. meliloti. The use of bacterial mutants has shown that the bacterial genes nodA, nodB and nodC, induced in response to plant factors, are essential for the production of a soluble factor(s) which act(s) to initiate root hair curling and pericycle cell divisions in Bradurhizobium sp. (Parasponia).
Russell et al. (1985) J. Bacteriol. 164:1301-1308 report the isolation of DNA regions encoding nodulation functions in strains of B. japonicum. The isolated DNA region was reported to show strong homology to nod regions of R. meliloti and R. leguminosarum, and to functionally complement a Nod- mutation in R. fredii. No sequence or transcript mapping of the cloned DNA was provided.
The precise biochemical role of the nod genes and their products in nodule development is unknown. Attempts to isolate nod gene mRNA and protein products from freeliving Rhizobium have been unsuccessful (Kondorosi et al. (1984). Protein products of nod genes have, however, been obtained by fusion of nod genes to strong E. coli promoters (Schmidt et al. (1984) EMBO J. 3:1705-1711; John, M. et al. (1985) EMBO J. 4:2425-2430) or in an E. coli in vitro transcription/translation system (Downie et al. (1985) Mol. Gen. Genet. 198:255-262). Schmidt et al. (1984) report the expression in E. coli minicells of several polypeptides encoded in the R. meliloti common nod region. Three polypeptides of 23, 28.5 and 44 kd, respectively, were mapped to the nod gene cluster. The 44 kd protein maps to a region of DNA strongly conserved among fast-growing rhizobia. John et al. (1985) supra identified the 44 kd protein as the product of the nodC gene. A fourth polypeptide product of 17.5 kd is mapped to the region of the nodD gene in R. meliloti. Downie et al. (1985) supra report the production of the presumptive nod gene products of R. leguminosarum by an in vitro translation/transcription system. Four polypeptides having molecular weights of 48, 45, 36 and 34 kd were reported to be the products of the nod genes. The 34 kd and 36 kd polypeptides are described as originating from a single gene and are reported to be the products of nodD.
Because establishment of nitrogen-fixing nodules is a multistage process involving coordinated morphological changes in both bacterium and plant, it is expected that the rhizobial nodulation genes are under precise regulatory control.
It has been suggested that an exchange of signals between plant and bacterium is requisite for mutual recognition and coordination of the steps of infection and nodulation development (Nutum, P.S. (1965) in Ecology of Soil Borne Pathogens, F.K. Baker and W.C. Snyder (eds.), University Of California press, Berkeley, pp. 231-247; Bauer, W.D. (1981) Ann. Rev. Plant Phys. 32:407-449: and Schmidt, E.E. (1979) Ann. Rev. Microbiol. 33:355-376). For example, root exudates have been linked to control of nodulation. Exudates have been reported to both stimulate (Thornton (1929) proc. Royal Soc. B 64:481; Valera and Alexander (1965) J. Bacteriol. 89:113-139; Peters and Alexander (1966) Soil Science 102:380-387) and inhibit (Turner (1955) Annals Botany 19:149-160; and Nutman (1953) Annals Botany 17:95-126) nodulation by rhizobia.
Although there are many sites at which root hair curling and cell division can be seen to initiate, only a small percentage of these sites produce functional nodules (Calvert et al. (1984) Can. J. Bot. 62:2375-2384). Nodule number is also influenced by factors of both bacterial and plant origin. For example, there are plant mutants which fail to repress nodulation as is normally seen in wild-type plants, resulting in a "super-nodulation" phenotype (Carroll et al. (1985) Proc. Natl. Acad. Sci. (USA) 82:4162-4166). Similarly, bacterial mutants which are capable of nodule induction but fail to fix nitrogen, induce greater numbers of nodules on plant roots than their parent wild-type strain (Scott et al. (1982) J. Mol. Appl. Genet. 1:315-326). Biochemical analysis of these plant mutants (Gresshoff and Delves (1986) in Plant Gene Research Vol. III. A Genetic Approach to Plant Biochemistry. A.B. Blonstein and P.T. King (eds), Springer-Verlag, Wein) together with split root experiments using a variety of bacterial strains (Kosslak R.M. and Bohlool, B.B. (1984) Plant Physiol. 75(1):125-130 ) has led to the suggestion that the bacterium, on invasion, produces a signal which results in the production of a systemic inhibitor in the plant shoot which acts to repress further nodulation in the susceptible root zone.
Recently, the chemical factors in legume exudates that are responsible for stimulation of nod gene expression in Rhizobia have been identified. EPO Publication No. 0245931, 1987 identified a structural related class of molecules, certain substituted flavones and flavanones as nod gene inducing factors. Individual purified molecules, either isolated from clover exudates or available from commercial sources, were found to induce nod gene expression. U.S. Pat. Application 035,516 filed Apr. 7, 1987, now abandoned discloses the use of chemical factors, i.e. flavonoids as inducers of nod genes of Bradyrhizobium japonicum. See also Kosslak, R.M. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7428-7432 which reported daidzein and genistein as the major components in soybean root exudate responsible for inducing the nod genes in B. japonicum.
In R. meliloti and R. leguminosarum, nodD is reported to be necessary in addition to plant factors for expression of the nodABC genes (Downie et al. (1985) Mol. Gen. Genet. 198:255-262; Mulligan and Long, (1985)Proc. Natl Acad. Sci 82:6609-6613; Rossen et al. (1985) EMBO J. 4:3369-3373. More recently, Shearman et al. (1986) EMBO J. 5:647-652, have reported that nodD is also required, in addition to plant factors for induction of nodF. Similarly, in R. trifolii, the nodABC genes are unable to confer root hair curling that is prerequisite for nodulation in the absence of the nodD gene (Schofield, Ph.D. Thesis (1984) Australian National University, Canberra). These results indicate that the nodD regulates the expression of other nod genes. The mechanism by which nodD regulates the expression of other nod genes is not yet known, but may involve the initial interaction of nodD directly or indirectly with legume exudate factors followed by binding of a product of nodD to DNA sequences in the promoter regions of the legume exudate inducible nod genes. In slow-growing rhizobia as well, e.g. in B. japonicum and Bradyrhizobium. sp. (Parasoonia), nodD has a similar regulatory function. See Nieuwkoop et al. (1987) J. Bact. 169:2631-2638; Scott (1986) Nucl. Acids Res. 14:2904-2919; and U.S. Pat. Applications 875,296 and 061,848, filed June 11, 1989, now allowed.
A highly conserved nucleotide sequence has been described in the promoter regions of several legume exudate inducible nod genes. This sequence precedes the R. trifolii nodABC and nodFE genes and the R. meliloti nodABC genes (Schofield and Watson (1986) Nucl. Acids Res. 14:2891-2904). The sequence has also been identified in the promoter region of the nodABC genes in the slow-growing Bradyrhizobium sp. (Parasponia) Scott (1986) Nucl. Acids Res. 14:2905-2919). More recently, the rhizobial nod consensus sequence has been described as t'. . . ATCCAYNNYNNYGYRGATGNWYKYKATCSAAWCAATCRATTTTACCARWYYKNSRR . . . 3'where N is A or G or C or T, Y is C or T, R is A or G, W is A or T, K is G or T and S is C or G. This sequence is believed to function in the regulation of expression of nod genes by chemical factors in legume exudate (Scott (1986) supra.
Scott (1986) supra describes and provides a a sequence for an open reading frame designated as nodK in Bradyrhizobium sp. (Parasponia) in the region between nodD and nodABC. This region is not present in fast-growing Rhizobia. Scott reports that computer analysis shows that nodK is as likely to be translated as nodA, nodB and nodC. Nieuwkoop et al. (1987) J. Bact. 169:2631-2638 report and provide a sequence for a similar open reading frame in R. japonicum having 30% homology to the Bradyrhizobium sp. (Parasponia) nodK.
While mutagenesis of host plants has yielded mutants with accelerated nodulation and increased nodule number, mutagenesis of bacteria has until now only yielded mutants which have altered host range, fail to nodulate, or are delayed in nodulation.
Burn, J. et al. (1987), "Four classes of mutations in the nodD gene of Rhizobium leguminosarum biovar viciae that affect its ability to autoregulate and/or activate other nod genes in the presence of flavonoid inducers," Genes and Development 1:456-464, report deficient ability to nodulate by R. leguminosarum in which nodABC were constitutively expressed.
Applicants know of no previous reports of the functionality of the nodK gene product, nor of the ability of insertions in this region, preferably insertions comprising constitutive promoters, to enhance nodulation and yield in host plants.